Cells interact with structurally distinct types of extracellular matrix in different tissues, at different stages of embryonic development, and during adult wound repair. This project focuses on addressing the following major questions concerning the mechanisms of these cell-extracellular matrix interactions: 1. What are the differences in cell adhesive structures and biological responses to 2D versus 3D matrices, as well as between different types of 3D matrices characteristic of different in vivo microenvironments? 2. What signal transduction mechanisms control cell behavior in different 3D microenvironments? We explored whether classical models of cell motility and signaling established using regular 2D cell culture are valid in the structurally complex 3D environments found in tissues. We discovered a unique mode of 3D migration using high-resolution live-cell imaging to visualize intracellular signaling using different in vitro models of 3D extracellular matrix. Primary dermal fibroblasts migrating in dermal tissue explants and cell-derived matrix were found to use blunt, cylindrical protrusions termed lobopodia, named after an intracellular pressure-driven protrusion used by migrating amoeba. In contrast, cells migrating in 3D collagen gels exhibited lamellipodia-based migration similar to 2D cell culture, with small, fan-shaped protrusions enriched in F-actin at the leading edge. A central feature of our approach was to measure polarization of Rho family GTPase signaling and the PI 3-kinase product PIP3. Classically, Rac1, Cdc42, and PIP3 are strongly polarized to the leading edge of cells migrating on 2D substrates; these signals are thought to regulate actin polymerization in lamellipodia at the leading edge of motile cells and help to determine directionality of migration. Using live-cell imaging with fluorescence-based biosensors, we compared the localization of these various signaling systems in primary human fibroblasts migrating in different 3D matrix environments. Active Rac1, Cdc42, and PIP3 were all polarized towards the leading edge during lamellipodia-based migration in 3D collagen. During lobopodia-based migration, however, polarization of Cdc42, Rac1, and PIP3 signaling was lost. Instead, signaling was concentrated in focal clusters behind leading protrusions, along the sides, and at the rear of the cell. Reducing actomyosin contractility switched the cells to lamellipodia-based 3D migration. A fruitful collaboration with Nria Gavara and Richard Chadwick in the NIDCD IRP helped to reveal that these modes of 3D migration were regulated by physical properties of the matrix. Modifying cell-derived matrix by limited proteolysis changed its elastic behavior, rendering it non-linearly elastic and transitioning the mode of 3D cell motility to lamellipodia-based migration. Conversely, inducing linear elastic behavior in the matrices by crosslinking 3D collagen or proteolyzed cell-derived matrix triggered lobopodia-based migration. Thus, the relative polarization of intracellular signaling identifies two distinct modes of 3D cell migration governed intrinsically by actomyosin contractility, and extrinsically by the elastic behavior of the 3D extracellular matrix. These novel findings also indicate that lamellipodia are not necessary for efficient 3D motility and that, contrary to dogma, polarized Rac1, Cdc42, and PIP3 signaling is not required for directionally persistent fibroblast migration. In order to explore how a 3D extracellular matrix regulates mechanotransduction to control the mode of leading-edge protrusion, we are investigating the function of intracellular actomyosin machinery during lamellipodia- and lobopodia-based migration. We are also exploring the mechanistic basis of the specific signaling responses of human fibroblasts to 3D extracellular matrix of differing biochemical compositions.